Study Of SIRT1 Gene Silencing On The Regulation Of Lipid Metabolism In Cultured Hepatocytes Of Dairy Cows In Vitro

Purpose Silent information regulator 1(SIRT1)plays a key role in the regulation of liver lipid metabolism.SIRT1 can also be activated in oxidative stress.Therefore,this study explored the mechanism of SIRT1 action in the development of fatty liver disease in dairy cows,laying a theoretical and experimental basis for elucidating the pathogenesis of fatty liver disease in dairy cows and finding therapeutic targets.Methods The experiment method was conducted in two aspects: 1)Cell culture to investigate the mechanism of high NEFAs on oxidative stress and lipid metabolism dysfunction in dairy cows,and 2.4 mM NEFAs,10 mM NAC,2.4 mM NEFAs,and 10 mM NAC were added in vitro.Cell supernatants and proteins were collected after 9 h of treatment.The biochemical kits were used to detect lipid metabolism(TG,TC,VLDL).The expression levels of SIRT1,PGC-1α,and SREBP-1c was determined by western blotting.The expression levels of SIRT1,PGC-1α,and SREBP-1c was determined by quantitative real-time polymerase chain reaction(qRT-PCR).2)To investigate the mechanism of SIRT1 on hepatocyte oxidative stress and lipid metabolism dysfunction in dairy cows,culture of primary hepatocytes of dairy cows for 72 h in vitro,the adenovirus(MOI=25,50,100,200)was added and the optimal MOI value was detected by confocal laser.Adenovirus infected the primary hepatocytes of dairy cows with multiplicity of infection(MOI=100)as Ad group,while negative control group(NC group)(viral vector without SIRT1 gene)and null control(K group)were established.The cells were infected with Ad-GFP-SIRT1 recombinant adenovirus and the expression levels of SIRT1,PGC-1α,PPARα,RXRα,SREBP-1c,ACCα,p-ACCα,FAS,ApoE and LDLR proteins were detected by western blotting.The mRNA expression of SIRT1,PGC-1α,PPARα,RXRα,SREBP-1c,ACCα,FAS,ApoE and LDLR were measured by qRT-PCR.Lipid metabolism indicators(TG,TC,VLDL)were detected by biochemical kits.Results1.After OA modeling,compared with the normal group,the content of TG and TC increased significantly in hepatocytes(P <0.05),and the content of VLDL did not increase significantly.The mRNA and protein expression of SIRT1,PGC-1α were significantly reduced(P <0.01),and the mRNA and protein expression of SREBP-1c were significantly increased(P <0.01).2.Compared with control group,the expression of SIRT1 protein was significantly decreased in NEFA group(P <0.05),the expression of PGC-1α protein was decreased,and the expression of SREBP-1c protein was significantly increased(P <0.05).Compared with the 2.4 mM NEFAs group,the expression of SIRT1 and PGC-1α protein increased in the 2.4 mM NEFAs+10 mM NAC group,and the expression of SREBP-1c protein significantly decreased(P <0.01).Compared with the control group,SIRT1 and PGC-1α mRNA expression levels decreased in the 2.4 mM NEFAs,and SREBP-1c mRNA expression levels significantly increased(P <0.01).Compared with the 2.4 mM NEFAs group,The mRNA expression of SIRT1 and PGC-1α was significantly increased in the 2.4 mM NEFAs +10 mM NAC group(P <0.05),and the expression of SREBP-1c mRNA was significantly decreased(P <0.01).3.Compared with the control group,after SIRT1 adenovirus transfection,SIRT1 and downstream regulation of lipid oxidation PGC-1α,PPARα,and RXRα protein expression was significantly reduced(P <0.05),SREBP-1c,ApoE,and LDLR protein expression was significantly increased(P <0.05),and the expression of FAS and ACCα protein was significantly increased(P <0.01).Compared with the control group,SIRT1,PGC-1α,and PPARα mRNA levels were significantly decreased(P <0.01),RXRα mRNA levels were significantly decreased(P <0.05),and SREBP-1c and FAS mRNA expression levels involved in lipid synthesis were significantly increased(P <0.01),ACCα mRNA expression was significantly elevated(P <0.05),and the mRNA expression levels of ApoE and LDLR,which regulate lipid transport,significantly increased(P <0.01).Conclusion The above results showed that hepatic lipid deposition was induced by oxidative stress and the SIRT1 gene was silenced by recombinant adenovirus technology.The accumulation of hepatic fat was promoted by regulating the SIRT1-SREBP-1c/ PGC-1α pathway.